Fluorescence in situ hybridization detection of chromosome 7 and/or 17 polysomy as a prognostic marker for cholangiocarcinoma

Cholangiocarcinoma (CCA) is highly endemic in the Northeast Thailand. Recently, chromosome aberrations provided new insights into pathogenesis of CCA. Therefore, chromosome aberration might be used as a prognostic factor and therapeutic planning of this cancer. This aim of this study is to examine the correlation between an increase of chromosome 7 (C7) and/or 17 (C17) copy number variants (CNVs) with clinicopathological data and the overall survival time (OS) of CCA patients using fluorescence in situ hybridization (FISH) assays. C7 and C17 CNVs were examined using FISH form 157 formalin-fixed paraffin-embedded (FFPE) tissues of CCA patients from Khon Kaen, Thailand between 2011 and 2015. OS was visualized using Kaplan–Meier plot. Univariate and multivariate analyses were used to determine the ability of the clinicopathological parameters to predict OS. C17 > trisomy (odd ratio, 6.944, P < 0.001), C7/17 trisomy (odd ratio; 4.488, P = 0.019), and C7/17 > trisomy (odd ratio; 6.723, P < 0.001) were independently predictive factors for lymph node metastasis. Interestingly, an increase of C7, C17, and C7/17 CNVs in both trisomy and > trisomy was independently correlated with short median OS. An increased of C7 and/or 17 have a potential as a poor prognostic marker in CCA patients.

Specimen preparation. Surgically resected specimens were processed by the standard formalin-fixed paraffin-embedding (FFPE) technique. The block of FFPE specimens were cut at 4 µm thickness for H&E staining. All H&E slides were marked the area of CCA by two experienced pathologists and then the slides were applied to construct tissue microarray (TMA) blocks. Tissue microarrays are compound paraffin blocks constructed by extracting cylindrical tissue core of 3 mm biopsies of marked area from all donor paraffin blocks which were re-embedded into a microarray or recipient block at defined array coordinates 38 . One slide of tissue microarray contains eight positive cases with CCA and one normal case control.
Fluorescence in situ hybridization assay. FISH technique was performed on TMA slides of FFPE tissues. The TMA specimens were sectioned at 4 µm thickness. The slides were deparaffinized at 60 °C overnight then immersed into xylene ambient 5 min 3 times each after that dehydrated with 100% ethanol 2 times 1 min each. The slides were pretreated with saline sodium citrate (SSC) buffer at 80 °C for 30-45 min depends on the condition of tissue, then rinsed with purified water for 3 min. Then, the slides were soaked in 50 ml protease buffer with 75 mg protease at 37 °C for 30-45 min. The slides were dehydrated in an ascending series (70-100% (v/v)) ethanol. The FISH centromere enumeration probes (CEP) were specific for pericentromeric regions of chromosomes 7 (C7) and 17 (C17) (Abbott ® , Laboratories Ltd; Illinois, USA). The probes preparation of one assay was mixed with hybridization buffer, probes and H 2 O, then, applied the probes mix to the target area on TMA slide, closed with coverslip and sealed with rubber cement. The hybridization step was performed on the ThermoBrite at 73 °C for 5 min followed by 37 °C 18 h. The first step of post-hybridization procedure, the slides were washed with 2X SSC/0.3% Tween 20 at ambient for 5 min and allowed coverslips to float off gently. Next, those slides were washed with 2X SSC/0.3% Tween 20 at 73 °C for 3 min. The last step, 4' ,6-diammidino-2 phenylindole (DAPI) was counterstained, closed with coverslip and storaged at − 20 °C protected from the light. Those slides were investigated under a fluorescence microscope (Olympus BX63; Tokyo, JAPAN) using GenASis FISH View & Spot Counting (Applied Spectral Imaging; Yokneam Illit, ISRAEL).
Evaluation criteria. We analyzed cut-off values based on FISH signals from 100 nuclei from each 10 FFPE tissues of surgical samples from cholangitis patients (n = 10). The mean value of C7 (% X for non-disomy) (a gain of more than two chromosomes within a single cell) was 4.5%, the upper cut-off value (%) was 8%, which was the mean + two standard deviations. While, the mean value of C17 (% X for non-disomy) (a gain of more two chromosomes within a single cell) was 4%, with the upper cut-off value (%) of 6%. We analyzed 50 sequential, non-overlapping, well-visualized, epithelial cell nuclei and consecutive area in all samples under a fluorescence microscope (Olympus BX63; Tokyo, JAPAN). The numerical chromosome aberration phenotypes of hepatectomy specimens were identified as disomy, trisomy, tetrasomy and > tetrasomy upon an increase of chromosomes as shown in Fig. 1. According to few tetrasomy, then " > trisomy" used as other representative group to statistical analysis. Polysomic cell was referred to an increase chromosome from those normal 2 CNVs. The number of counted cells was listed and the highest percentage of those phenotype was taken as the representative phenotype of individual patient. Each sample was independently examined by three investigators (RD, MT, and KI) who were unsighted to the clinicopathological data. If the assessments of the two investigators differed, a consensus was accomplished by discussion.

Statistical analyses.
Statistical analyses were carried out using the Statistical Package for the Social Sciences; SPSS software V.23.0 (IBM, Chicago, IL, USA). Comparison between signal copy numbers and the clin-  The correlation between C7 and C17 CNVs and clinicopathological features of CCA patients. As shown in Fig. 1, the chromosome phenotypes were defined as disomy, trisomy, and more than trisomy using FISH technique. The correlation between distribution patterns of C7 copy number and clinical features of CCA patients were examined and the results were summarized in Table 1. The incidence of C7 copy number variants (CNVs) was significantly higher in patients with higher AST level (P = 0.05). The difference of the incidence of CNVs in between disomy and trisomy group was as same as that in between trisomy vs. > trisomy (P = 0.031). In addition, the incidence of CNVs was significantly associated with the gross pathological types particularly). Disomy was significant difference from > trisomy. Patients who had gall bladder, lymph node and distant metastasis have greater proportion of both C7 and C17 > trisomy than those patients who had no evidence of gall bladder metastasis ( Fig. 3a-c). Moreover, the prevalence of C7 CNVs significantly correlated with distant metastasis (P < 0.001) ( Table 1). The incidence of C17 CNVs was significantly higher in patients with high AST level. There were significant differences in the incidence of disomy and trisomy as well as trisomy vs. > trisomy. The incidence of C17 CNVs was significantly associated with the gross pathologic types. The incidence of C17 CNVs also significantly correlated with gall bladder, lymph node and distant metastasis (P < 0.001) ( Table 2).
The correlation between clinicopathological factors, C7/C17 CNVs and metastatic features. Univariate and multivariate analyses were applied to evaluate the correlations between FISH results of C7/C17 CNVs and metastatic features including lymph node, gall bladder and distant metastasis and other clinicopathological factors including demographic data, some liver function data, histopathology in CCA patients (Tables 3 and 4).

Discussion
CCA is asymptomatic in the early stage and is usually diagnosed at the advanced stage resulting in unsatisfied outcome with poor prognosis in spite of various treatments 9 . Prognostication is important in clinical and ethical approaches of clinicians engaged in oncology and palliative care of CCA, because it helps clinicians for planning an appropriate therapeutic strategy for advanced cancer patients 17 . Prognostication is made mostly based on tumor staging 18,19 . In addition, several factors including chromosomal aberration 21,22 have been reported as prognostic factors for advanced cancer patients 20 . FISH assay is applied for several cancers including numerical and structural chromosome abnormalities in CCA patients 31 . In this study, we found that an increase of C7 CNVs is associated with high AST level, gross mass forming type and metastasis to lymph node, gall bladder and distant organs. These results indicate that metastatic status that reported potential pathological factors for predicting CCA recurrence 39 is highly relevant to C7 > trisomy in CCA patients. Furthermore, an increase of C17 CNVs is associated with the clinical parameters such as high AST level, mass forming type, lymph node, gall bladder and distant metastasis. Interestingly, an increase of C7 and C17 CNVs is associated with microscopic growth www.nature.com/scientificreports/ patterns particularly, mass-forming type which have the poorest prognosis in iCCA patients 40,41 as same as the metastatic features. AST is useful for the evaluation of bile duct and liver injury that shown a modest impact on the OS of the iCCA patients 42,43 . Thus, in case of poor prognostic iCCA that underwent C7 and C17 aberrations shown associated significantly with high AST level. However, For the mechanisms of association with high AST level and an increase of C7 and C17 CNVs, not yet elucidated.
In the univariate analyses, C7 > trisomy C17 > trisomy and mix C7/17 > trisomy were significantly associated with each metastasis type, to lymph node to gall bladder and to distant organ, respectively. However, in the multivariate analyses, an increase of C7 and C7/17 > trisomy was an independent predictive factor of lymph node metastasis only. Nevertheless, an increase of C17 CNV and C7/17 > trisomy were independent predictive factors of gall bladder metastasis. For distant metastasis, an increase of C17 and C7/17 CNVs were independent predictive factors. Thus, an increase of C7 and C17 strongly correlated with lymph node and distant metastasis that indicated advanced stage CCA followed by TMN and American Joint Committee on Cancer (AJCC)/Union for International Cancer Control (UICC) Staging Systems 44 .
Additionally, in univariate analysis, we found that patients with C7 > trisomy and trisomy have short median survival time than those C7 disomy. Similarly, patients with C17 > trisomy and trisomy have shorter median survival time than those with C17 disomy (OS 3.03 Vs.15.53 Vs.45.83). Moreover, pattientts having both C7 and 17 C7 > trisomy and trisomy showed short median survival time than those with disomy (OS 2.23 Vs.15.33 Vs.50.23), respectively. Thus, an increase of C7, C17 and C7/17 CNVs were significantly associated with poor prognosis in CCA patients in univariate analysis. Interestingly, an increase of C7, C17 copy numbers as trisomy and greater than trisomy were independently correlated with short median survival time by multivariate analysis. Similar to our results, Kato et al. (2008) reported that, using multivariate analysis, an increase of C7 copy number and advanced clinical stage were independently predictive of poor OS 32 . While trisomy C7 is usually associated  30 . In addition, human epidermal growth factor receptor 2 proto-oncogene (HER-2, also known as c-erbB2, ERBB2) that regulates cell growth, survival, differentiation and migration is located on C17 (q12-q21) 48 . ERBB2 is also the  www.nature.com/scientificreports/ most common genetic alterations in invasive breast carcinomas, associated with poor prognosis and response of the tumor to the ERBB2 monoclonal antibody trastuzumab in breast cancer 49 . Furthermore, ERBB receptors are frequently overexpressed in CCA leading to tumor progression and poor prognosis 50 . Besides, copy number analysis also detected more frequent ERBB2 amplification in liver fluke-related CCAs, which may have considerable clinical implications 40 . ERBB-2, EGFR and Met are members of tyrosine kinase growth factor receptors (TKGFRs) that play major roles in bile duct carcinogenesis. Thereby, overexpression of these genes as mentioned above resulting from an increase of C7 and C17 CNVs elicit an essential role in CCA progression 37,47 . Markedly, an increase of C7, C17 and C7/C17 copy numbers are strongly independent markers for the short survival time of CCA patients. Our study was performed on a large scale CCA specimens over 5 years retrospective research. Therefore, the results of FISH assay using FFPE samples significantly support its potential as prognostic marker of poor prognosis in CCA patient. In further study, we would add more probes or apply UroVysion ® probes in extended retrospective duration time.

Conclusion
An increase of C7, 17 and 7/17 CNVs in FFPE specimens were significant correlated with metastatic factors in CCA patients. In particular, having more than trisomy of C7 and 7/17 were statistically significant independent predictive factor of lymph node metastasis. Also, Patients who have more than trisomy of C17 and 7/17 were statistically significant independent predictive factor of gall bladder metastasis. Moreover, > trisomy of C7/17 was an independent predictive factor of organ metastasis. Patients having trisomy or > trisomy of either C7 or C17 have shorter median survival time than those having normal disomy. Thus, polysomy of both C7 and C17 have a potential as a poor prognostic marker of CCA patients.

Data availability
All data sets used and /or analyzed during the study are not publicly available due to personal or identifying data of patients but are available from the corresponding author on reasonable request.